1. Field of the Invention
The present invention relates to an immunizing composition and method for inducing an immune response against β-secretase cleavage site of amyloid precursor protein. The present invention further relates to antibodies raised or generated against the β-secretase cleavage site of amyloid precursor protein and the use thereof in passive immunization.
2. Description of the Related Art
Amyloid Precursor Protein and β-Secretase:
The extracellular deposition of short amyloid peptides in the brains of patients is thought to be a central event in the pathogenesis of Alzheimer's disease. Evidence that amyloid may play an important role in the early pathogenesis of AD comes primarily from studies of individuals affected by the familial form of AD (FAD) or by Down's syndrome. The generation of amyloid β peptide (Aβ) occurs via a regulated cascade of cleavage in its precursor protein, AβPP (amyloid precursor protein). At least three enzymes are responsible for AβPP proteolysis and have been tentatively named α, β, and γ secretase. The recent identification of several of these secretases is a major leap in understanding how these secretases regulate amyloid peptide formation. One of the main therapeutic goals is the inhibition of secretases that produce Aβ from the large precursor protein. The theoretical specificity and tractability of protease targets suggest that it should be possible to generate secretase-specific protease inhibitors that penetrate the blood brain barrier. Many studies using new knowledge of the ability of the β-secretase enzyme (BACE) to identify inhibitors by screening or rational design approaches are already underway (U.S. Pat. Nos. 5,744,346; 5,942,400; 6,221,645 B1; 6,313,268 B1; and published PCT applications WO 00/47618, WO 98/21589, and WO 96/40885). At this point, there is no evidence of additional functions of Aβ, so there are no serious concerns about reduction of this metabolite. Both β- and γ-secretases are present in many different cells in the body and it is reasonable to assume that they have substrates in addition to AβPP. Consequently, complete inhibition of one of these enzymes might result in toxicity problems, particularly under the chronic treatment conditions that would presumably be required. At the mRNA level, BACE is expressed widely in the human brain. Expression is also high in the pancreas, although enzymatic activity in this tissue is low. Apart from AβPP cleavage, it is not known if BACE possesses other activity and so it is too early to predict what toxicity β-secretase inhibitors may have.
Proteolytic processing of the amyloid precursor protein (AβPP) generates amyloid β (Aβ) peptide which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. To initiate Aβ formation, β-secretase cleaves AβPP at the N-terminus of Aβ to release APPsβ, an approximately 100-kD soluble N-terminal fragment, and C99, a 12-kD C-terminal fragment which remains membrane bound. The exact site of β-secretase cleavage has been determined (FIG. 1). Amyloid plaque Aβ starts at Aspl and this cleavage site is therefore of major interest. Cleavage by β-secretase at the amino terminus of the Aβ peptide sequence, between residues 671 and 672 of AβPP, leads to the generation and extracellular release of β-cleaved soluble AβPP, and a corresponding cell-associated carboxy-terminal fragment.
One of the familial AD families was shown to have a mutation in AβPP that coincided with the predicted cleavage site of β-secretase. This double mutation, first identified in a Swedish pedigree, was also found to mechanistically result in overproduction of Aβ peptide relative to wild sequence when it was transfected into cells, suggesting that it was a better substrate for the β-secretase enzyme. This prediction has recently been borne out to be true. A Met to Leu substitution at the Pl position of APP, found in the “Swedish” familial AD mutation which causes early-onset AD, dramatically enhances β-secretase cleavage, but many other substitutions (for example, Met to Val) decrease β-secretase cleavage. These findings demonstrated the presence of a β-secretase activity responsible for a cleavage event that liberated the N terminus of Aβ peptide and showed the process was secretory rather than lysosomal, the favored hypothesis at the time.
Blood Brain Barrier:
The blood-brain barrier (BBB) (Johansson, 1992; Ermisch, 1992; Schlosshauer, 1993) is formed by a monolayer of tightly connected microvascular endothelial cells with anionic charges. This layer separates two fluid-containing compartments: the blood plasma (BP) and extracellular fluid (ECF) of the brain parenchyma, and is surrounded by astroglial cells of the brain. One of the main functions of the BBB is to regulate the transfer of components between the BP and the ECF. The BBB limits free passage of most agent molecules from the blood to the brain cells.
In general, large molecules of high polarity, such as peptides, proteins, (e.g., enzymes, growth factors and their conjugates, oligonucleotides, genetic vectors and others) do not cross the BBB. Therefore poor agent delivery to the CNS limits the applicability of such macromolecules for the treatment of neurodegenerative disorders and neurological diseases.
Several delivery approaches of therapeutic agents to the brain circumvent the BBB. Such approaches utilize intrathecal injections, surgical implants (Ommaya, 1984 and U.S. Pat. No. 5,222,982) and interstitial infusion (Bobo et al., 1994). These strategies deliver an agent to the CNS by direct administration into the cerebrospinal fluid (CSF) or into the brain parenchyma (ECF).
Drug delivery to the central nervous system through the cerebrospinal fluid is achieved by means of a subdurally implantable device named after its inventor, the “Ommaya reservoir”. The reservoir is used mostly for localized post-operative delivery of chemotherapeutic agents in cancers. The drug is injected into the device and subsequently released into the cerebrospinal fluid surrounding the brain. It can be directed toward specific areas of exposed brain tissue which then adsorb the drug. This adsorption is limited since the drug does not travel freely. A modified device developed by Ayub Ommaya, whereby the reservoir is implanted in the abdominal cavity and the injected drug is transported by cerebrospinal fluid (taken from and returned to the spine) all the way to the ventricular space of the brain, is used for agent administration.
Diffusion of macromolecules to various areas of the brain by convection-enhanced delivery is another method of administration circumventing the BBB. This method involves: a) creating a pressure gradient during interstitial infusion into white matter to generate increased flow through the brain interstitium (convection supplementing simple diffusion); b) maintaining the pressure gradient over a lengthy period of time (24 hours to 48 hours) to allow radial penetration of the migrating compounds (such as: neurotrophic factors, antibodies, growth factors, genetic vectors, enzymes, etc.) into the gray matter; and c) increasing drug concentrations by orders of magnitude over systemic levels. Through their direct infusion into the brain parenchyma, the site-specific biomolecular complexes of U.S. Pat. No. 6,005,004 deliver the agent to neuronal or glial cells, as needed, and be retained by these cells. Moreover, the site-specific complexes containing neuronal targeting or internalization moieties are capable of penetrating the neuronal membrane and internalizing the agent.
Another strategy to improve agent delivery to the CNS is by increasing the agent absorption (adsorption and transport) through the BBB and their uptake by the cells (Broadwell, 1989; Pardridge et al., 1990; Banks et al., 1992; and Pardridge, edited by Vranic et al., 1991. The passage of agents through the BBB to the brain can be enhanced by improving either the permeability of the agent itself or by altering the characteristics of the BBB. Thus, the passage of the agent can be facilitated by increasing its lipid solubility through chemical modification, and/or by its coupling to a cationic carrier, or still by its covalent coupling to a peptide vector capable of transporting the agent through the BBB. Peptide transport vectors are also known as BBB permeabilizer compounds (U.S. Pat. No. 5,268,164).
Phage Display:
Combinatorial phage display peptide libraries provide an effective means to study protein:protein interactions. This technology relies on the production of very large collections of random peptides associated with their corresponding genetic blueprints (Scott et al, 1990; Dower, 1992; Lane et al, 1993; Cortese et al, 1994; Cortese et al, 1995; Cortese et al, 1996). Presentation of the random peptides is often accomplished by constructing chimeric proteins expressed on the outer surface of filamentous bacteriophages such as M13, fd and f1. This presentation makes the repertoires amenable to binding assays and specialized screening schemes (referred to as biopanning (Parmley et al, 1988)) leading to the affinity isolation and identification of peptides with desired binding properties. In this way peptides that bind to receptors (Koivunen et al, 1995; Wrighton et al, 1996; Sparks et al, 1994; Rasqualini et al, 1996), enzymes (Matthews et al, 1993; Schmitz et al, 1996) or antibodies (Scott et al, 1990; Cwirla et al, 1990; Felici et al, 1991; Luzzago et al, 1993; Hoess et al, 1993; Bonnycastle et al, 1996) have been efficiently selected.
Filamentous bacteriophages are nonlytic, male specific bacteriophages that infect Escherichia coli cells carrying an F-episome (for review, see Model et al, 1988). Filamentous phage particles appear as thin tubular structures 900 nm long and 10 nm thick containing a circular single stranded DNA genome (the +strand). The life cycle of the phage entails binding of the phage to the F-pilus of the bacterium followed by entry of the single stranded DNA genome into the host. The circular single stranded DNA is recognized by the host replication machinery and the synthesis of the complementary second DNA strand is initiated at the phage ori(−) structure. The double stranded DNA replicating form is the template for the synthesis of single-stranded DNA circular phage genomes, initiating at the ori(+) structure. These are ultimately packaged into virions and the phage particles are extruded from the bacterium without causing lysis or apparent damage to the host.
Peptide display systems have exploited two structural proteins of the phage; pIII protein and pVIII protein. The pIII protein exists in 5 copies per phage and is found exclusively at one tip of the virion (Goldsmith et al, 1977). The N-terminal domain of the pIII protein forms a knob-like structure that is required for the infectivity process (Gray et al, 1981). It enables the adsorption of the phage to the tip of the F-pilus and subsequently the penetration and translocation of the single stranded phage DNA into the bacterial host cell (Holliger et al, 1997). The pIII protein can tolerate extensive modifications and thus has been used to express peptides at its N-terminus. The foreign peptides have been up to 65 amino acid residues long (Bluthner et al, 1996; Kay et al, 1993) and in some instances even as large as full-length proteins (McCafferty et al, 1990; McCafferty et al, 1992) without markedly affecting pIII function.
The cylindrical protein envelope surrounding the single stranded phage DNA is composed of 2700 copies of the major coat protein, pVIII, an α-helical subunit which consists of 50 amino acid residues. The pVIII proteins themselves are arranged in a helical pattern, with the α-helix of the protein oriented at a shallow angle to the long axis of the virion (Marvin et al, 1994). The primary structure of this protein contains three separate domains: (1) the N-terminal part, enriched with acidic amino acids and exposed to the outside environment; (2) a central hydrophobic domain responsible for: (i) subunit:subunit interactions in the phage particle and (ii) transmembrane functions in the host cell; and (3) the third domain containing basic amino acids, clustered at the C-terminus, which is buried in the interior of the phage and is associated with the phage-DNA. pVIII is synthesized as a precoat protein containing a 23 amino acid leader-peptide, which is cleaved upon translocation across the inner membrane of the bacterium to yield the mature 50-residue transmembrane protein (Sugimoto et al, 1977). Use of pVIII as a display scaffold is hindered by the fact that it can tolerate the addition of peptides no longer than 6 residues at its N-terminus (Greenwood et al, 1991; Iannolo et al, 1995). Larger inserts interfere with phage assembly. Introduction of larger peptides, however, is possible in systems where mosaic phages are produced by in vivo mixing the recombinant, peptide-containing, pVIII proteins with wild type pVIII (Felici et al, 1991; Greenwood et al, 1991; Willis et al, 1993). This enables the incorporation of the chimeric pVIII proteins at low density (tens to hundreds of copies per particle) on the phage surface interspersed with wild type coat proteins during the assembly of phage particles. Two systems have been used that enable the generation of mosaic phages; the “type 8+8” and “type 88” systems as designated by Smith (Smith, 1993).
The “type 8+8” system is based on having the two pVIII genes situated separately in two different genetic units (Felici et al, 1991; Greenwood et al, 1991; Willis et al, 1993). The recombinant pVIII gene is located on a phagemid, a plasmid that contains, in addition to its own origin of replication, the phage origins of replication and packaging signal. The wild type pVIII protein is supplied by superinfecting phagemid-harboring bacteria with a helper phage. In addition, the helper phage provides the phage replication and assembly machinery that package both the phagemid and the helper genomes into virions. Therefore, two types of particles are secreted by such bacteria, helper and phagemid, both of which incorporate a mixture of recombinant and wild type pVIII proteins.
The “type 88” system benefits by containing the two pVIII genes in one and the same infectious phage genome. Thus, this obviates the need for a helper phage and superinfection. Furthermore, only one type of mosaic phage is produced.
The phage genome encodes 10 proteins (pI through pX) all of which are essential for production of infectious progeny (Felici et al, 1991). The genes for the proteins are organized in two tightly packed transcriptional units separated by two non-coding regions (Van Wezenbeek et al, 1980). One non-coding region, called the “intergenic region” (defined as situated between the pIV and pII genes) contains the (+) and the (−) origins of DNA replication and the packaging signal of the phage, enabling the initiation of capsid formation. Parts of this intergenic region are dispensable (Kim et al, 1981; Dotto et al, 1984). Moreover, this region has been found to be able to tolerate the insertion of foreign DNAs at several sites (Messing, 1983; Moses et al, 1980; Zacher et al, 1980). The second non-coding region of the phage is located between the pVIII and pIII genes, and has also been used to incorporate foreign recombinant genes as was illustrated by Pluckthun (Krebber et al, 1995).
Immunization with Phage Display:
Small synthetic peptides, consisting of epitopes, are generally poor antigens requiring the chemical synthesis of a peptide and need to be coupled to a large carrier, but even then they may induce a low affinity immune response. An immunization procedure for raising anti-AβP antibodies, using as antigen the filamentous phages displaying only EFRH peptide, was developed in the laboratory of the present inventor (Frenkel et al., 2000 and 2001). Filamentous bacteriophages have been used extensively in recent years for the ‘display’ on their surface of large repertoires of peptides generated by cloning random oligonucleotides at the 5′ end of the genes coding for the phage coat protein (Scott and Smith, 1990; Scott, 1992). As recently reported, filamentous bacteriophages are excellent vehicles for the expression and presentation of foreign peptides in a variety of biologicals (Greenwood et al., 1993; Medynski, 1994). Administration of filamentous phages induces a strong immunological response to the phage effects systems (Willis et al., 1993; Meola et al., 1995). Phage coat proteins pIII and pVIII discussed above are proteins that have been often used for phage display. The recombinant filamentous phage approach for obtaining specific peptide antigens has a major advantage over chemical synthesis, as the products obtained are the result of the biological fidelity of translational machinery and are not subject to the 70-94% purity levels common in the solid-phase synthesis of peptides. The phage presents an easily renewable source of antigen, as additional material can be obtained by growth of bacterial cultures.
Immunization with the EFRH (SEQ ID NO:2) epitope displaying phage may, in a short period of time, raise the high concentration of high affinity (IgG) antibodies able to prevent the formation of β-amyloid and to minimize further toxic effects. The level of antibody in the sera was found to be related to the number of peptide copies per phage (Frenkel et al., 2000b).
The antibodies resulting from EFRH (SEQ ID NO:2) phage immunization are similar regarding their immunological properties to antibodies raised by direct injection with whole amyloid β (Table 1). These antibodies recognize the full length Aβ-peptide (1-40) and exhibit anti-aggregating properties as antibodies raised against whole Aβ peptide and/or amyloid β (Frenkel et al., 2000b, 2001). The high immunogenicity of filamentous phages enables the raising of antibodies against self-antigens. Immunization of guinea pigs with EFRH (SEQ ID NO:2) phage as an antigen, in which the Aβ peptide sequence is identical to that in humans, resulted in the production of self-antibodies (Frenkel et al., 2001).
TABLE 1Competitive inhibition by various peptides within Aβ of serum antibodyraised against f88-EFRH compared to amyloid anti-aggregating antibody*.MICEanti-aggregatingPEPTIDERESIDUESSERUMantibody*.FRH(residues 4-6 of Aβ)~10−3M3 × 10−3MEFRH(residues 3-6 of Aβ; SEQ ID N0: 2)6.0 × 10−6M3 × 10−6MDAEFRH(residues 1-6 of Aβ; residues 1-6 of SEQ ID NO: 3)3.0 × 10−6M8 × 10−7MDAEFRHD(residues 1-7 of Aβ; residues 1-7 of SEQ ID NO: 3)5.0 × 10−6M9 × 10−7MDAEFRHDSG(residues 1-9 of Aβ; SEQ ID NO: 3)5.0 × 10−6M1 × 10−6MAβ(1-40)3.0 × 10−6M8 × 10−7MWVLD(SEQ ID NO: 4)Nd **Nd ***Frenkel et. al. 1998** IC50 value of less than 10−2 M which cannot be detected by ELISA assay.
The above data demonstrated that a recombinant bacteriophage displaying a self-epitope can be used as a vaccine to induce autoantibodies for disease treatment. Filamentous phages are normally grown using a laboratory strain of E. coli, and although the naturally occurring strain may be different, it is reasonable to assume that delivery of phage into the gut will result in infection of the natural intestinal flora. The laboratory of the present inventor has found that UV inactivated phages are as immunogenic as their infective counterparts. There is evidence of long lasting filamentous phages in the guts of the immunized animals that may explain the long lasting immune response found in pIII immunized mice (Zuercher et al., 2000).
Due to the high antigenicity of the phage, administration can be given by the intranasal route, which is the easiest way for immunization without any use of adjuvant. As olfactory changes are proposed to play a role in Alzheimer's disease (Murphy, 1999) mucosal immunization is an effective induction of specific Aβ IgA antibodies for preventing local pathologic effect of the disease.
The efficacy of phage-EFRH antigen in raising anti-aggregating β-amyloid antibodies (Solomon and Frenkel, 2000) versus whole β-amyloid shows that:
a. the high immunogenicity of the phage enables production of high titer of IgG antibodies in a short period of weeks without need of adjuvant administration;
b. self-expression of the antigen led to long-lasting immunization;
c. the key role of the EFRH epitope in β-amyloid formation and its high immunogenicity led to anti-aggregating antibodies which recognize whole β-amyloid peptide, substituting the use of β-amyloid fibrils.
Antibody Engineering:
Antibody engineering methods were applied to minimize the size of mabs (135-900 kDa) while maintaining their biological activity (Winter et al., 1994). These technologies and the application of the PCR technology to create large antibody gene repertoires make antibody phage display a versatile tool for isolation and characterization of single chain Fv (scFv) antibodies (Hoogenboom et al., 1998). The scFvs can be displayed on the surface of the phage for further manipulation or may be released as a soluble scFv (˜25 kd) fragment.
The laboratory of the present inventor engineered an scFv which exhibits anti-aggregating properties similar to the parental IgM molecule (Frenkel et al., 2000a). For scFv construction, the antibody genes from the anti-AβP IgM 508 hybridoma were cloned. The secreted antibody showed specific activity toward the AβP molecule in preventing its toxic effects on cultured PC 12 cells. Site-directed single-chain Fv antibodies are the first step towards targeting therapeutic antibodies into the brain via intracellular or extracellular approaches.
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